RNAi-Mediated Tocopherol Deficiency Impairs Photoassimilate Export in Transgenic Potato Plants

Abstract

Tocopherols (vitamin E) are lipophilic antioxidants presumed to play a key role in protecting chloroplast membranes and the photosynthetic apparatus from photooxidative damage. Additional nonantioxidant functions of tocopherols have been proposed after the recent finding that the Suc export defective1 maize (Zea mays) mutant (sxd1) carries a defect in tocopherol cyclase (TC) and thus is devoid of tocopherols. However, the corresponding vitamin E deficient1 Arabidopsis mutant (vte1) lacks a phenotype analogous to sxd1, suggesting differences in tocopherol function between C4 and C3 plants. Therefore, in this study, the potato (Solanum tuberosum) ortholog of SXD1 was isolated and functionally characterized. StSXD1 encoded a protein with high TC activity in vitro, and chloroplastic localization was demonstrated by transient expression of green fluorescent protein-tagged fusion constructs. RNAi-mediated silencing of StSXD1 in transgenic potato plants resulted in the disruption of TC activity and severe tocopherol deficiency similar to the orthologous sxd1 and vte1 mutants. The nearly complete absence of tocopherols caused a characteristic photoassimilate export-defective phenotype comparable to sxd1, which appeared to be a consequence of vascular-specific callose deposition observed in source leaves. CO 2 assimilation rates and photosynthetic gene expression were decreased in source leaves in close correlation with excess sugar accumulation, suggesting a carbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity. This conclusion is further supported by an increased photosynthetic capacity of young leaves regardless of decreased tocopherol levels. Our data provide evidence that tocopherol deficiency leads to impaired photoassimilate export from source leaves in both monocot and dicot plant species and suggest significant differences among C3 plants in response to tocopherol reduction.

Tocopherols Play a Crucial Role in Low-Temperature Adaptation and Phloem Loading in Arabidopsis

“…The maize sxd1 carbohydrateaccumulation phenotype was intriguing in that it suggested an unexpected link between the tocopherol pathway and primary carbohydrate metabolism, although the mechanism involved was unclear. A similar carbohydrate phenotype did not occur in the orthologous Arabidopsis vte1 mutant but was observed in VTE1 RNA interference (RNAi) knockdown lines in potato (Solanum tuberosum) (Hofius et al, 2004).…”

“…Previous studies of the maize sxd1 mutant and potato VTE1-RNAi lines (both affecting tocopherol cyclase activity) had suggested a linkage between carbohydrate metabolism and tocopherol biosynthesis, as in both cases carbohydrates accumulated to high levels in mature leaves at normal growth temperatures (19 to 308C) (Russin et al, 1996;Provencher et al, 2001;Hofius et al, 2004). The absence of this phenotype in Arabidopsis vte1 and vte2 at 228C raised questions regarding the universality of any interaction between tocopherol synthesis and carbohydrate metabolism (Figures 6 and 7, day 0).…”

“…Structural Changes in Low-Temperature-Treated Tocopherol-Deficient Mutants Previously, callose was reported to accumulate at the bundle sheath/vascular parenchyma interface of the maize sxd1 mutant and in vascular tissue of potato VTE1-RNAi lines, both of which are defective in tocopherol cyclase (Botha et al, 2000;Hofius et al, 2004). To determine whether callose deposition also occurs in Col, vte2-1, and vte1-1, leaves were harvested at 0, 1, 3, and 13 d of 7.58C treatment and aniline blue-positive fluorescence was assessed.…”

Genetic Control of Carbon Partitioning in Grasses: Roles of Sucrose Transporters and Tie-dyed Loci in Phloem Loading

“…It is not understood how a lack of tocopherol leads to ectopic callose deposition. However, this function is conserved in potato (Solanum tuberosum) and Arabidopsis (Arabidopsis thaliana), as plants deficient for the Sxd1 orthologous gene show a similar callose-deposition and carbohydrateaccumulation phenotype (Hofius et al, 2004;Maeda et al, 2006).…”

Alterations in Tocopherol Cyclase Activity in Transgenic and Mutant Plants of Arabidopsis Affect Tocopherol Content, Tocopherol Composition, and Oxidative Stress

“…Interestingly, the corresponding maize mutant, sxd1, shows a much more severe phenotype than the Arabidopsis vte1 plant, because anthocyanins, sugars, and starch strongly accumulate in sxd1 leaves (Russin et al, 1996;Provencher et al, 2001). Similarly, down-regulation of VTE1 expression in transgenic potato resulted in reduced growth and accumulation of carbohydrates (Hofius et al, 2004).…”